中文摘要：

目的建立一种有效分离小鼠肝肿瘤细胞的原代培养方法,为肝癌发生发展的机制研究提供有效的实验手段。方法以9月龄H-ras12V转基因肝癌小鼠为实验材料,采用传统的小鼠肝细胞原代培养的灌注分离法,并对其进行改进,即在灌注的同时,用剪刀剪去肿瘤表面阻塞的血管和组织,疏通灌注液流,达到有效消化肿瘤组织的目的。对用此改良灌注法分离的肝肿瘤细胞和肿瘤周围组织细胞进行成活率计数,并进行后续的原代细胞培养,观察第3、5、7天的细胞生长状态和形态特征。结果通过对传统的灌注分离肝细胞法进行改良,分离的肝肿瘤细胞成活率由未改良前的10%提升到40%。原代培养的结果表明,分离的肝肿瘤细胞与肝肿瘤周围的正常肝细胞的形态和随时间延长的凋亡变化状态没有显著差异。结论改良的灌注法能够有效的分离小鼠肝肿瘤细胞,并显著提高其存活率和细胞质量,可用于后续的原代细胞培养并开展相关的实验。另外,本文对改良灌注法的详细叙述也为相关的科研工作者提供了切实可行的操作规程。

英文摘要：

To estabolish an effective primary culture method for mouse liver tumor cells and provide an effective experimental method for the researches on mechanismes of hepatic tumorigenesis. 9 month-old H-rasl2V transgenic liver cancer mouse model was used. Liver tumor cells was isolated based on the improved traditional liver perfusion method, briefly, cut off the blocking arteries and tissues on the surface of the liver tumor to dredge the perfusion liquid for achieving the effective digestion of tumor tissues. The cell viability of per-tumor tissues and tumor tissues were counted, purified and cultured. The morphological characteristics of cells were observed every day until 14 days. Compared to the traditional liver perfusion method, the improved method elvated the viability of cells from 10% to 40%. The primary culture results showed that there was no significant difference between cells isolated from peri-tumor tissues and tumor tissues on the morphological characteristics and apoptosis ratio with elongated culture time. The improved traditional liver perfusion method is an effective method to isolate the hepatic tumor cells and significantly improved the viability and quality of the cells which is enough for subsequent experiments. Furthermore, the detailed protocol descripted in this article provides a practicable and feasible operating instruction for related researchers.